During the current reporting period we have focused on the following projects: 1) Studies of a New Autoinflammatory Disease Caused by Mutations in RIPK1 Members of two families presented with a previously undescribed autoinflammatory disorder characterized by early-onset periodic fever episodes and severe intermittent lymphadenopathy. In some individuals this disease was selectively responsive to therapeutic IL-6 inhibition with tocilizumab. Exome sequencing revealed that RIPK1 was the only gene in which a variant from both families satisfied filtering criteria. In one family the sole affected individual had a de novo heterozygous mutation, p.Asp324Asn, while the second family demonstrated 3-generation inheritance of the p.Asp324His heterozygous mutation in 5 affected individuals. These two mutations result in different substitutions at the same critical residue that is specifically required for RIPK1 cleavage by caspase-8. This aspartate is highly conserved across species, and both mutations are absent from variant databases. The mutations impaired RIPK1 cleavage by caspase-8 as well as by caspase-6 in vitro, though cleavage in human cells appeared to be mediated predominantly by caspase-8. There are multiple possible mechanisms by which cleavage-resistant RIPK1 could lead to inflammatory pathology. Patient-derived cells showed a modest but significant increase in NF-kappaB activation compared to control cells. This rare monogenic disease resulting from mutations in RIPK1 demonstrates the importance of caspase-mediated RIPK1 cleavage in vivo for maintaining human inflammatory homeostasis. A manuscript describing these findings has been submitted for publication. 2) Continuation of Studies of the Natural History of HA20 In collaboration with extramural physicians, a total of 16 patients (13 female) from 7 families with a genetic diagnosis of HA20 were included. The disease commonly manifested in early childhood (range: first week of life to 29 years of age). The main clinical symptoms were recurrent oral, genital, and/or gastrointestinal ulcers (16/16), musculoskeletal (9/16) and gastrointestinal complaints (9/16), cutaneous lesions (8/16), episodic fever (7/16), and recurrent infections (7/16). Clinical phenotypes varied considerably, even within families. Relapsing-remitting disease course was most common, and one patient died. Laboratory abnormalities included elevated acute-phase reactants and fluctuating presence of various autoantibodies such as antinuclear antibodies (4/10 patients tested) and anti-dsDNA (2/5). Tissue biopsy of different sites revealed nonspecific chronic inflammation (6/12 patients tested), findings consistent with class V (membranous) lupus nephritis in one patient, and pustules and normal results in 2 patients each. All patients were treated: 4/16 received colchicine and 12/16 received various immunosuppressive agents. Cytokine inhibitors effectively suppressed systemic inflammation in 7/9 patients. From this study we concluded that early-onset recurrent oral, genital, and/or gastrointestinal ulcers are the hallmark feature of HA20, while the frequency and intensity of other clinical manifestations varied highly. In the paper summarizing these results, we recommended basing treatment on disease severity, with cytokine inhibitors often required to control relapses. A paper summarizing these findings was published in the Annals of the Rheumatic Diseases. 3) Continuation of Studies of Regulation of the NLRP3 Inflammasome Gain-of-function mutations in NLRP3 cause the spectrum of cryopyrin-associated periodic syndromes (CAPS), an important group of autoinflammatory disorders. The NLRP3 protein nucleates an inflammasome that mediates the maturation of the proinflammatory cytokine IL-1beta in response to a variety of perturbations of cellular physiology. The generation of reactive oxygen species (ROS) is one of the major factors known to activate the NLRP3 inflammasome, but the mechanisms by which changes in the cellular redox state regulate the NLRP3 inflammasome are not known. To identify an ROS-mediated regulator of NLRP3 inflammasome activation, we utilized anti-NLRP3 antibodies to precipitate immune complexes from the lysates of bone marrow derived macrophages (BMDMs) from wild-type (WT) or NLRP3-KO mice, and analyzed these immune complexes by mass spectrometry. We found that cofilin-1 is an important interacting protein. Cofilin-1 bound the nucleotide-binding domain (NBD) of NLRP3, and dissociated from NLRP3 when the cells were stimulated with known NLRP3 inflammasome activators, such as ATP or nigericin. These NLRP3 inflammasome activators generate ROS that induce cofilin-1 oxidation, leading to intramolecular disulfide bond formation between 2 cysteine residues at amino acids 39 and 80. This oxidation induces a conformational change of cofilin-1 and dissociation from NLRP3, which results in the activation of the NLRP3 inflammasome. Indeed, the assembly of NLRP3 inflammasome components was impaired and IL-1 release was significantly suppressed in BMDMs ectopically expressing oxidation-resistant mutant cofilin-1 (C39A or C80A). In addition, knockdown of cofilin-1 in LPS-primed BMDMs induced NLRP3 inflammasome activation without activator treatment. We also observed that the interaction of cofilin-1 with CAPS-associated mutant NLRP3 proteins was substantially diminished relative to WT NLRP3, which was associated with constitutive activation of the NLRP3 inflammasome. To examine the role of cofilin as a redox sensor for NLRP3 inflammasome activation in vivo, we generated knock-in (KI) mice expressing oxidation-resistant mutant cofilin-1 (C39A or C39S). Unexpectedly, the IL-1beta release from the BMDMs of the KI mice was higher than WT BMDMs when the cells were stimulated with ATP after LPS priming. Moreover, serum IL-1beta levels of KI mice after intraperitoneal injection of lipopolysaccharide (LPS) were significantly higher than WT mice. KI mice express very low levels of mutant cofilin-1, which may account for the increased IL-1beta release. Indeed, similar to the results with cofilin-1 knockdown, LPS-primed KI BMDMs release substantial amounts of IL-1beta without activators. Taken together, these results suggest that cofilin-1 is an important negative regulator of the NLRP3 inflammasome, and is a molecular link between ROS and NLRP3 inflammasome activation. A manuscript describing these results will be prepared during the upcoming reporting period. 4) Neutrophil Biology in PAPA Syndrome PAPA is caused by mutations in PSTPIP1, which encodes a pyrin-binding protein. Patients develop severe neutrophil-mediated inflammation of the skin and joints. We provided samples from 12 PAPA patients to NIAMS collaborators. Their studies demonstrated that neutrophils from PAPA patients exhibited increased potential to form neutrophil extracellular traps (NETs), relative to controls. In addition, PAPA sera exhibited impaired NET degradation. NETs from PAPA neutrophils stimulated IL-6 release from healthy control macrophages. Overall, these studies suggest an important role for neutrophils and NET formation in PAPA. A manuscript describing the results of this study was published in the Annals of the Rheumatic Diseases. 5) Studies of the Role of the Pyrin Inflammasome in PAPA We have conducted studies of human-derived cell lines to investigate the pathophysiology of PAPA syndrome. In an overexpression study, differentiated U-937 cells ectopically expressing PAPA-associated mutant PSTPIP1 released approximately 8 times as much IL-1beta as cells expressing WT PSTPIP1. IL-1beta release was substantially decreased by knocking down MEFV, but not by knocking down NLRP3, AIM2, or NLRC4, suggesting that the pyrin inflammasome plays an important role in IL-1beta production in PAPA.